Biological indicator

ABSTRACT

A biological indicator for monitoring the effectiveness of a sterilization or disinfection treatment or process. The biological indicator comprises a substrate having a surface layer containing functional groups thereon desirably free of silicon linking groups. The functional groups are added to the surface through the use of a functionalized silane coupling agent, wherein two different functional groups are generally utilized and the functionalized silane is applied in a uniform and consistent manner thereby assuring that the microorganism or etiological agent indicators are attached to the substrate in a uniform and consistent manner through non-covalent bonding to the functional groups. After being subjected to sterilization or other similar disinfecting treatments or processes along with various articles such as instruments, the indicator can be cultivated or cultured to determine the effectiveness of the sterilization or disinfection treatment or process.

CROSS REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 12/082,642, filed Apr. 11, 2008 for BIOLOGICALINDICATOR, now U.S. Pat. No. 7,741,107 which is a continuation of U.S.patent application Ser. No. 11/135,719, filed May 24, 2005, and now U.S.Pat. No. 7,416,883 issued Aug. 26, 2008 which is hereby fullyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a biological sterile indicator for monitoringsterility or disinfection, etc., having a carrier surface containingfunctional groups which are covalently bonded to one or moremicroorganisms through a crosslinking reagent. The indicator desirablycontains a uniform and consistent microorganism population which can beutilized in evaluating various sterilizing treatments of medical devicesand accessories, instruments, solutions, surfaces, clothes, and thelike, to indicate the degree or success of sterilization with respect toarticles that are to be reused or terminally sterilized.

2. Description of the Prior Art

Biological indicators are used to test and/or determine theeffectiveness of sterilization processes. A typical biological indicatorcontains a calibrated population of microorganisms having a highresistance to the sterilization process under investigation. Afterexposure to the sterilization process, the biological indicators areincubated in a culture medium to encourage growth of any remainingviable microorganisms. Self-contained biological indicators contain theculture medium within the indicator, typically in a frangible vial.Spore strip biological indicators are combined with a separate containerof culture medium after the monitored sterilization process. Subsequentmicrobial growth, as demonstrated by a color change or turbidity of thegrowth medium, is an indication that the sterilization process wasineffective.

Bacterial spores are typically favored for biological indicators due tothe fact that microbial spores are accepted as being more resistant tosterilization processes than most other types of microorganisms, andthus it is assumed that a sterilization process that will kill microbialspores will also kill any other contaminating microorganism.

The choice of bacterial spores is dependent upon the sterilization mode,sterilization type, and technique to be evaluated. For example,Geobacillus stearothermophilus spores are used to monitor sterilizationsystems employing moist heat, peracetic acid, hydrogen peroxide, andother peroxy compounds in both the liquid and vaporous state becausethese indicator spores are highly resistant to oxidative chemistries.Similarly, Bacillus subtilis spores are employed to monitor ethyleneoxide sterilization, and dry heat sterilization systems.

The microorganisms are generally supported on a carrier or substrate,such as a strip or disk. The substrate is formed from a material whichis compatible with the sterilization process and does not containadditives which may influence the sterility assessment. Materials suchas filter paper, chromatography paper, blotter paper, glass fibers,polymer plastics, ceramics, stainless steel, and metaloxide articles areoften used as a substrate or carrier.

To distribute the organisms on the substrate, a suspension ofmicroorganisms in water or alcohol is conventionally pumped to a needlewhich is suspended over a web of the paper or other substrate material.The paper is moved under the needle at a constant rate, causing a trailof suspension to form on the paper as it passes beneath the needle.Alternatively, the suspension is manually transferred by use of amicropipette to the substrate. The web of impregnated paper is then cutto the appropriate size for use as the indicator, typically as teststrips or test disks.

As well as the necessity for a controllable quantity of themicroorganism to the substrate surface, it is important that themicroorganism be sufficiently immobilized on the surface of thesubstrate. Various types of linkages of the microorganism to thesubstrate surface have been proposed, including hydrophobic andelectrostatic interactions, ion exchange, and van der Waal's forces.U.S. Pat. No. 4,478,946, relates to the adsorption of nonfunctionalproteins to a surface and the employment of crosslinking agents tocovalently attach functional proteins to adsorbed non-functionalproteins. However, such immobilization techniques often provide lessthan desirable attachment, especially in aqueous environments. U.S. Pat.No. 5,077,210 relates to active agents such as proteins covalentlyimmobilized on substrates carrying hydroxyl groups. A silane is bound tothe substrate and coupled to a heterobifunctional crosslinker at onefunctional group leaving a free functional group, different than thefirst group, to which a protein is bound while retaining high proteinfunctionality. The silane has a functional group which reacts with thehydroxyl group of the substrate and a thiol terminal group which reactswith a functional group of a heterobifunctional crosslinking agent whichcontains a succinimide group that reacts with an amino group of theactive agent.

SUMMARY OF THE INVENTION

A biological indicator for determining whether a sterilization,disinfection, or other such biocidal treatment was effective with regardto complex organic living organisms, comprises a substrate inherentlycontaining functional groups thereon or added thereto, or aself-assembled surface layer thereon such as a functional groupcontaining monolayer (SAM). The functional groups include hydroxylgroups, halide groups, amine groups, and the like, and desirably excludethio groups. In a preferred embodiment, a crosslinking agent such as aheterobifunctional compound is utilized which provides a covalentlinkage of a microorganism indicator such as a spore, vegetativeorganism, or an etiological agent to the functionalized substratesurface. In such an embodiment, the microorganism indicator is tightlybound to either the surface layer or preferably to the substrate nothaving any surface layer thereon, and is very difficult to remove bywashing, fluid turbulence, and the like.

In another embodiment, the inherent functional hydroxyl groups of asubstrate such as glass can be utilized. In this embodiment, themicroorganism indicator or etiological agent is bound to the hydroxylfunctional surface as by way of, but not limited to, a physical,electronic, etc. manner or through hydrogen bonding, but not by acovalent bond.

In a less desired embodiment, a functional group is added to thesubstrate surface through the use of an organic silane coupling agentwherein two different functionalized groups are generally utilized. Inthis embodiment, the microorganism indicator is bound to the silanecoupling agent as through a physical, electronic, etc. manner, but notby a covalent bond.

In either embodiment, the functional agent is applied in a uniform andconsistent manner thereby ensuring that the microorganism populationthereon will also be uniform and consistent.

The biological indicators are generally utilized to indicate whether asterilization, disinfection, or other such biocidal treatment of variousarticles such as surgical instruments have been effective so that thearticles can be reused.

A biological indicator for monitoring sterility; comprising: asubstrate; a surface layer containing functional end groups residing onsaid substrate, said surface layer being substantially free of anysilicon linking atoms; a microorganism indicator; and a crosslinkingreagent covalently bonded to said surface layer functional groups andcovalently bonded to said microorganism indicator.

A biological indicator, comprising: a substrate and optionally a surfacelayer residing on said substrate, said substrate or said optionalsurface layer containing functional groups; an etiological agentcomprising a bioterrorism agent, a clinically relevant organism, aresistant strain of bacteria, or a subcellular constituent, orcombinations thereof; and a crosslinking agent covalently bonded to saidsubstrate functional group or to said optional surface layer functionalgroup and covalently bonded to said etiological agent.

DESCRIPTION OF THE INVENTION

A biological indicator is described that is suitable for monitoringand/or determining the biocidal effectiveness of a sterility,disinfection, or deactivation process of microorganisms, Prions,etiological agents, and the like, which are generally highly resistantto such treating processes. While spores, e.g. endospores, are thepreferred test microorganism because they generally have a highresistance to many sterile processes, other microorganisms includingfungi, mycobacteria, vegetative bacteria, and protozoa, can also be usedin place of spores. Desirably, the microorganisms have sulfur end groupsthereon and if not, they can be thiolated to contain sulfur end groups.Examples of endospores include Geobacillus stearothermophilus, Bacillussubtilis, Bacillus subtilis globigii, Clostridium sporogenes, Bacilluscereus, and Bacillus circulans. Examples of fungi include Aspergillusniger, Candida albicans, Trichophyton mentagrophytes, and Wangielladermatitis. Examples of mycobacteria which can be utilized in thepresent invention include Mycobacterium chelonae, Mycobacteriumgordonae, Mycobacterium smegmatis, and Mycobacterium terrae.

Examples of vegetative bacteria include Aeromonas hydrophila,Enterococcus faecalis, Streptococcus faecalis, Enterococcus faecium,Streptococcus pyrogenes, Escherichia coli, Klebsiella (pneumoniae),Legionella pneumophila, Methylobacterium, Pseudomonas aeruginosa,Salmonella choleraesuis, Helicobacter pylori, Staphylococcus aureus,Staphylococcus epidermidis, and Stenotrophomonas maltophilia. Withrespect to vegetative bacteria, vegetative cells and or theirconstituent parts can be affixed to a solid support matrix by the samemechanism(s) and survive drying and storage by means of deposition inthe presence of one or more of a variety of excipients. Excipients are abroad class of generally inert compounds that are used to stabilizelabile entities. A subclass of excipients includes the carbohydrates andnotably oligo and polymeric saccharides. One example of such a compoundis the disaccharide trehalose. High concentrations of trehalose in thetissues of certain organisms allow them to survive in a state of waterdeficiency and have also been shown to revive functional cellularcomponents after dehydration. In fact, trehalose is known to providestability to membranes and other macromolecular structures essential tothe viability of a cell under extreme environmental conditions (i.e.freeze drying). Other stabilizing excipient compounds include (but arenot limited to) simple sugars, e.g. sucrose, glucose, maltose, longchain polymers e.g. dextrans, starch, agarose and cellulose. Othernon-carbohydrate based excipients might also include proteins,phosphonates, buffering agents, waxes, lipids, oils or other hydrocarbonbased materials.

Examples of protozoa include Giardia lamblia and Cryptosporidium parvum.

Spores are preferred in the present invention because they are highlyresistant to sterilization, etc., processes.

In addition to monitoring and/or determining the effectiveness ofvarious treatments or processes with regard to sterilizing, etc.endospores, fungi, etc., the indicators of the present invention canalso be utilized with regard and/or determining the effectiveness of asterile or disinfecting treatment or process.

Etiological Agents and Other Non-simulative Agents

Etiological agents include bioterrorism agents, clinically relevantagents, sub-cellular components, and emerging resistant strains ofbacteria. In addition to the simulative organisms selected on the basisof their acceptance as representing a ‘most resistant’ nature (e.g.Geobacillus stearothermophilus) and other microorganisms set forthabove, non self-replicating agents and sub cellular components orproducts of cells can be selected based on their clinical significanceor because of their use as agents of bioterrorism. These organisms areoften comprised of strains which may now have resistance to normal meansof antibiotic treatment or chemical disinfection due to natural ormanmade modifications. Examples of the former type minimally includeVRE's (Vancomycin Resistant Enterococci), MSRAs (Methicillin ResistantStaphylococcus aureus), and Mycobacterium cheloni. These clinicallysignificant organisms are particularly of interest because the VRE's andMRSAs represent organisms which have developed resistance to theirtypical therapeutic countermeasures (antibiotic resistance) and M.cheloni exemplifies organisms that have developed resistance to someheretofore effective modes of disinfection (glutaraldehyde resistance).There are also a number of emerging etiological agents of significancefor which there may not yet be a simulative alternative, which mayrepresent a special risk or challenge to therapeutic course of action ordisinfection. One such example is prions. Prions are not livingorganisms, per se, but their function as disease causing agents arerelated to their structure and this structure/function relationship canbe employed to determine their relative infectivity. Othernon-autonomous agents (e.g. viruses) as well as sub cellular elementsand proteinaceous prions are contemplated in the present invention.

Etiological agents which can be used as bioterrorism agents or diseasesinclude anthrax (Bacillus anthracis), botulisum (Clostridium botulinumtoxin), brucella species (brucellosis), Burkholderia mallei (glanders),Burkholderia pseudomallei (Melloidosis), Chlamydia psittaci(psittacosis), cholera (Vibrio cholerae), Clostridium perfringens(Epsilon toxin), Coxiella burnetii (Q fever), emerging infectiousdiseases such as nipah virus and hantavirus, Escherichia coli O157:H7(E. Coli), food safety threats (e.g. salmonella species), Francisellatularensis (tularemia), plague (Yersinia pestis), ricin toxin fromRicinus communis (castor beans), Rickettsia prowazekii (typhus fever),Salmonella typhi (typhoid fever), shigella (shigellosis), smallpox(Variola major), Staphylococcal enterotoxin B, Vibrio cholerae(cholera), Viral encephalitis (alphaviruses [e.g. Venezuelan equineencephalitis, eastern equine encephalitis, western equineencephalitis]), viral hemorrhagic fevers (filoviruses [e.g. Ebola,Marburg] and arenaviruses [e.g. Lassa, Machupo]), water safety threats(e.g. Cryptosporidium parvum), and Yersinia pestis (plague), orcombinations thereof.

The test microorganism can be a single species or a combination ofspecies. Where a combination of species is utilized, for typicalsterilization processes for medical devices, the preferred organismswill most often be a combination of Geobacillus stearothermophilus andBacillus subtilis.

A solid substrate is preferably utilized as the spore carrier material.The substrate can be any inorganic material such as silicon includingcrystalline silicon; various types of glasses including soda-lime,borosilicate glass, phosphate glass, borophosphate glass,boroaluminosilicate glass, and the like having any shape or form such asa sheet, fiber, bead, ballotini; various ceramics which can be definedas earthly raw materials in which silicon and its oxide and complexcompounds known as silicates occupy a predominate portion and which havebeen heated to high temperatures such as structural clay productsincluding tile and terra cotta, various porcelains, porcelain enamels,and the like; metal such as palladium, platinum, iron, copper, gold;various inorganic substrates containing metalized surfaces such as thoseimmediately set forth, or various metal oxides of groups 4 through 14 ofthe Periodic Table including titanium oxide, zirconium oxide, ironoxide, copper oxide, aluminum oxide, silica such as quartz, sapphire,and the like.

Another group of substrates which can be utilized are various organiccompounds including cellulose in various forms such as paper, filterpaper, cardboard, and the like. Various polymers can be exemplified by,but not limited to, acrylic polymers including acrylic acid and acrylatepolymers, various polyolefins such as polyethylene and polypropylene,polyvinyl alcohol polymers; polystyrene; and the like. The above notedsubstrates can also be composites of the above noted compounds.

The substrates can be electronically and/or physically modified byplasma, electron beams, gamma radiation, photo activation, and the like.Such treatments can use existing functional groups, add functionalgroups, or make assessable inherent groups (i.e. active), on thesubstrate surface such as hydroxyl groups. Normally, at least part ofthe exposed surface of the substrate will be planar, although curvedsurfaces can be treated in accordance with the present invention; e.g.,the substrate surface can be formed on the inside, or outside surface ofa test tube or from a multi-well plate or the outside of a bead orcontainer. While the substrate is preferably solid, it can be partiallyporous or porous.

The substrates can exist in a large variety of forms so long as theyprovide an exposed surface. Examples of suitable forms include fibers,wires, wafers, discs, sheets, microscope slides, crystallizing dishes,closed absorption cells, glass media ampoules, and the like. Preferredsubstrates include various forms of cellulose such as paper, glassfibers, alkaline earth aluminoborosilicate glass, polystyrene, and thelike.

The substrates of the present invention can naturally or inherentlycontain functional groups thereon, e.g. various glasses often containhydroxyl groups or amine groups. Alternatively, a separate surface layercontaining functional groups can reside or exist on the substrate as inthe form of a monolayer such as a SAM (self-assembled monolayer method),which is well known to the art and to the literature. The inherent oradditional layer of functional groups comprise hydroxyl, amine,carboxylic acid, carbonyl, various halides such as chlorine or bromine,and various alkenes containing a total of from about 2 to at least about20 carbon atoms. Thio groups are generally avoided.

It is an important aspect of the present invention to utilize variousdifferent types of crosslinking agents to covalently bond themicroorganism indicator to the substrate. The crosslinking agent canhave functional groups which are the same or different and numeroustypes of functional groups exist and may fall into more than onecategory such as various amine compounds including primary, secondary,and tertiary amines, various imines, various imides including aniline,imidyl esters of carboxylic acids, hydroxyl, carboxylic acids, alkenylgroups having from 2 to at least about 20 carbon atoms, halides such aschlorine or bromine, nitroaryl halides, alkoxy groups having a total offrom 1 to at least about 20 carbon atoms, anhydrides, aldehydes, cyanos,various sulfur containing groups such as thios, disulfides or dithiosand the like. Preferred reactive end groups of the crosslinking agentsgenerally include the various amines with primary amines beingpreferred, thio or other sulfur containing groups, carboxyl, andhydroxyl. Common compounds containing amines therein includesuccinimidyl esters, maleimides, azides, and iodoacetamides.

Suitable crosslinking reagents include homobifunctional crosslinkingreagents, heterobifunctional crosslinking reagents, trifunctionalcrosslinking reagents, zero-length crosslinking reagents, andphotoreactive crosslinking reagents. Heterobifunctional crosslinkingreagents are preferred.

Examples of homobifunctional crosslinking reagents include variousamines such as Bis[sulfosuccinimidyl] suberate (BS3), and3-[2-aminoethyldithio] propionic acid HC1 (AEDP).

Examples of heterobifunctional crosslinking reagents includeN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),N-sulfosuccinimdyl6-[3′-(2-pyridyldithio)-propionamido] hexanoate(Sulfo-LC-SPDP), N-succinimidyl 6-[3′-(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP), N-succinimidyl acetylthioacetate (SATA),N-succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate(SMCC), N-sulfosuccinimidyltrans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (Sulfo-SMCC),N-Succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-Sulfosuccinimidyl(4-iodoacetyl) aminobenzoate (Sulfo-SIAB),m-Maleimidobenzoyl-N-hydroxysuccimide ester (MBS), Succinimidyl4-[p-maleimidophenyl] butyrate (SMPB), Sulfosuccinimidyl4-[p-maleimidophenyl] butyrate (Sulfo-SMPB), N-(a-Maleimidoacetoxy)succinimide ester (AMAS),Succinimidyl-6-[β-maleimidopropionamido]hexanoate (SMPH), N-Succinimidyliodoacetate (SIA), N-κ-Maleimidoundecanoic acid (KMUA), and Succinimidyl3-[bromoacetamido] propionate (SBAP). Other crosslinking agents includeN-Hydroxysuccinimide (NHS), N-Hydroxysulfosuccinimide (Sulfo-NHS),3-[2-Aminoethyldithio] propionic acid HCl (AEDP) (can also be ahomobifunctional crosslinking reagent), Methyl N-succinimidyl adipate(MSA), N-β-Maleimidopropionic acid (BMPA), N-[κ-Maleimidoundecanoicacid]-hydrazine (KMUH), and N[β-Maleimidophenyl propionic acid]hydrazide TFA (BMPH), and N[p-Maleimidophenyl] isocyanate (PMPI).

An example of a trifunctional crosslinking reagent is Tris-succinimidylaminotriacetate (TSAT).

Examples of zero-length crosslinking reagents include1-Ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride (EDC).

Examples of photoreactive crosslinking reagents (i.e. react specificallywith available nucleophiles upon UV illumination) include aminereactives such as 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidylester (ATFB, SE); 4-azido-2,3,5,6-tetrafluorobenzoic acid STP ester,sodium salt (ATFB, STP ester); Benzophenone-4-isothiocyanate;4-benzoylbenzoic acid, succinimidyl ester;N-5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOS); Sulfosuccinimidyl2-[m-azido-o-nitrobenzamide]ethyl-1,3′-dithiopropionate (SAND);N-Succinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate (SANPAH);Sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate(Sulfo-SANPAH); and Succinimidyl-[4-(psoralen-8-yoloxy)]butyrate (SPB);thiol reactives include N-([2-pyridyldithio]ethyl)-4-azidosalicylamide;and Benzophenone-4-maleimide; and carbonyl reactives include4-azido-2,3,5,6-tetrafluorbenzylamine hydrochloride.

Still other crosslinking reagents include N-gamma-maleimidobutyryloxysuccinimide ester, 1,3-bismaleimido propane which has the formula

the N-hydroxysuccinimide ester ofN-(4)-carboxycyclohexylmethyl)-maleimide, and the N-hydroxysuccinimideester of 3(2-pyridyl-dithio)-propionic acid having the formula

The various homobifunctional, heterobifunctional, trifunctional,zero-length, and photoreactive crosslinking reagents will thus reactthrough one end group with the substrate or separate surface layerfunctional compound to form a covalent bond therewith whereas theremaining one or more functional end group(s) will react with amicroorganism indicator or etiological agent and also form a covalentbond therewith. The order of reaction is not important since thecrosslinking agent can first be reacted with a functional group of thesurface or of a surface layer and subsequently with the microorganism,or initially with the microorganism and subsequently with the surface orsurface layer functional group.

It is an important aspect of the invention to bond the microorganismindicator to the substrate through strong covalent bonds of at leastabout 10, desirably from about 10 to about 150, and preferably fromabout 25 to about 125 kilocalories per mole. Such strong bonds oranchorages of the microorganism indicator or etiological agent to thesubstrate are resistant to adverse or turbulent conditions associatedwith washer-disinfectors and/or liquid chemical sterile processes. Inother words, the biological indicators of the present invention arehighly resistant to being washed away or separated from the substrate asfrom jet spray impingement, turbulent hydrostatic forces, and the like.

The biological indicator of the present invention is prepared byapplying a layer of crosslinking reagents to the substrate or to thesubstrate-separate layer laminate containing a functional group thereon.Preferably, the functional groups are substantially free of or containno silicon linking atoms. Silicon linking atoms occur when afunctionalized silane coupling agent is utilized. By substantially freeit is meant that generally less than about 10%, desirably less thanabout 5% and preferably less than about 2 or 3%, or nil, that is none,of the functional groups are bonded to the substrate through anintermediate silicon atom. The substrates are also substantially free ofproteins, partially hydrolyzed proteins and peptides. The amount of anysuch proteins, etc. if they exist is low such as generally less thanabout 10%, desirably less than about 5% and preferably less than about2% or 3% of the functional groups which are bonded to the substrate.Depending upon the type of the above noted functional groups containedon the substrate or the surface layer, a crosslinking reagent isutilized having at least one end group which readily reacts therewithand forms a covalent bond. For example, if the substrate surface has anamine functional group, one of the functional groups of the crosslinkingreagent such as a heterobifunctional reagent will readily reacttherewith such as an aldehyde, an imide, a carboxyl group, an amidylester of a carboxylic acid, an anhydride, and the like. One such methodrelates to utilizing the crosslinking reagent in a buffer such as aphosphate buffer at relatively neutral conditions, for example a pH offrom about 6 to about 8, for a suitable amount of time to permit acovalent bond to be formed with the functional group of the substrate.Excess crosslinking reagent can be removed in any conventional manner asby rinsing or washing, or the like.

The remaining one or more end groups of the crosslinking reagent is afunctional group that readily reacts with the microorganism indicator,or etiological agent and forms a covalent bond therewith. Thus, themicroorganism indicator, etiological agent, or disease causing agent orstimulant, etc., are covalently bonded to the substrate functional groupthrough the crosslinking reagent. Such functional groups, as notedabove, that react with the microorganism include a halogen, a melaminederivative, a thio group, a maleimide, a malamide, etc. Should theremaining functional group of the crosslinking reagent be blocked as bya pyridine group, the same can be removed by utilizing a common reducingagent. Examples of suitable reducing agents include dithiothreitol(DTT), β-mercaptoethanol, DMF, DMSO, lithium aluminum hydride, sodiumborohydride, and the like. In a preferred embodiment of the presentinvention, the heterobifunctional agent is SPDP which upon reduction ofthe compound removes the pyridine group leaving a hydrogen sulfide groupwhich readily reacts and covalently bonds to the sulfur present in thethio group of the spore thereby immobilizing the spore to the substratesurface.

Since the preferred microorganism of the present invention is abacterial spore, and since the spore generally contains sulfur endgroups thereon, it exemplifies a desired embodiment of the invention.Should the spore or microorganism not contain a sulfur end group, it canbe thiolated before reaction with the crosslinking agent. Thiolation ofmicroorganisms is known to the art and to the literature.

The various one or more crosslinking reagents can be reacted in avariety of ways such as at room temperature, at elevated temperatures,by radiation such as ultraviolet light, and the like.

An important aspect of the present invention is the uniform and/orconsistent population of microorganism indicators or etiological agentson the substrate such that a low standard deviation is or combinationsthereof; attained such as generally about 50% or less, desirably about25% or less, and preferably about 10% or less based upon one area, forexample, a cm² as compared to another area on the substrate. The uniformdistribution of a microorganism indicator or etiological agent isprepared as by suspending a selected spore microorganism such as in anaqueous solution, generally water or solvent. Solvents can includeethanol, methanol, and other alcohols, with ethanol being the preferred.It should be understood, however, that the spores can be suspended in awide variety of solutions, so long as the viability and resistanceproperties of the spores are not compromised.

Alternatively, the above-noted crosslinking reagents can be initiallyreacted with one or more types of microorganism indicators, oretiological agent, and the like, and then subsequently applied in anaqueous solution or solvent to a functional group containing substratewhereupon the crosslinking agent is covalently bonded thereto.

The prepared substrate having either an inherent functional group orfunctional groups provided by a self-assembled monolayer (SAM) which iscovalently bonded to a crosslinking reagent is then inoculated with amicroorganism, such as a spore suspension at a predetermined, particularconcentration. The concentration of the spore suspension will vary,depending on the application requirements and desired rate ofapplication to the substrate, but will generally be from about 10⁴cfu/ml to about 10⁹ cfu/ml. Inoculation of the spore solution onto thecarrier is accomplished by immersing or dipping the surface into thespore solution, pipetting, spraying, or printing a fixed volume ofsuspension onto the substrate. The actual amount of microorganismdeposited or residing on the biological indictor will be from about 10⁴to about 10⁷ cfu/biological indicator. Where the substrate is generallyflat such as a closed adsorption cell or microscope slide, the entiresurface of the carrier is covered. Where the substrate is generallycurved surface, such as a glass media ampoule, only an end portion orthe entire surface is covered.

The inoculated substrate is then dried at ambient temperatures of fromabout 17° C. to about 25° C. for a period of time from about 1 min toabout 30 min. or longer, or at elevated temperatures to reduce dryingtimes. Following inoculation of the carrier, the carrier is rinsed withwater or a solvent to remove spores that are not tightly bonded to thesurface. The substrate may then be inspected visually utilizingphoto-optimetric instruments such as an optical or scanning probemicroscope to determine uniformity of the microorganism indicatorpopulation on the carrier surface. The population of the carrier surfacecan also be enumerated by titering macerated or sonicated samples, or byother means known to those skilled in the art.

In lieu of microorganism indicators such as spores, various etiologicalagents can be applied in a similar manner to the substrate to achieve adesirable concentration on the surface.

Due to the fact that substrates or surface layers can be made havinguniform and consistent concentrations of functional groups thereon whichare then covalently bonded via the crosslinking reagent to themicroorganisms or etiological agents, they are strongly adhered to thesubstrate with very little loss, if any, due to turbulent and/orchemical sterile treatments or processes. The microorganisms such as thespores or etiological agent thus serve as a very effective biologicalindicator as to the effectiveness of the sterilization of variousarticles. For example, the biological indicators of the presentinvention are most useful with liquid chemical-type sterilizationprocesses but can also be utilized in sterilizing processes such assteam, chemical, radiation, vapor phase, etc. for sterilizing variousarticles set forth below. Upon completion of the sterilizing cycle, theone or more biological indicators are incubated in a manner well knownto the art and to the literature. If any of the microorganisms, forexample spores, or etiological agents have survived the sterilizingprocess, they will grow during incubation under the appropriateincubation conditions known to those skilled in the art. The presence ofany growth is an indication that the sterilization cycle may not havebeen successful. Thus, after incubation of the biological indicators,the disinfection or degree of sterility is determined in theconventional manner. The end result is often determined by the nature ofthe articles being sterilized with a log reduction of at least fromabout 4 to about 12, and preferably at least about 5 or about 6 to about8 or about 9. A log reduction of 6 means that one or less microorganismsin 1,000,000 remain following exposure to a sterilization process.

The type of microorganism or spore, or etiological agent utilized in thebiological indicator can often be the same as the specific organismsought to be destroyed. For example, with regard to biological warfareagents, if a composition or a container is thought to contain anthrax,an anthrax spore can be utilized so upon completion of the sterilizationprocess, it can be determined whether or not the process was effectivein destroying the anthrax indicator.

Articles which can be subject to sterilization utilizing a biologicalindicator of the present invention are numerous and comprise instrumentsincluding surgical instruments, equipment including tubing as for thetransportation of medical and pharmaceutical compounds, compositionssuch as various powders, mixtures, solutions, cloth, and whenever anindication of a sterilizing or disinfecting process is sought.

The function and advantage of the embodiments of the present inventionwill be more fully understood from the examples below. The followingactual examples are intended to illustrate the benefits of the presentinvention, but do not exemplify the full scope of the invention.

EXAMPLE 1A

A substrate is utilized containing an amine terminated surface, whethermanufactured with the functionality such as a glass or a polystyrenesubstrate, or modified using a self-assembled monolayer with the desiredend group or treated by some physical or chemical means to release ormake accessible functional groups. A heterobifunctional crosslinkingreagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), isreacted to the amine terminated surface in phosphate buffered saline for30-60 minutes (a 20 mM SPDP stock solution is made in either DMSO orEtOH) at temperatures preferably between 17° C.-40° C. All excess SPDPis removed by rinsing in phosphate buffer. The surface is then reducedusing dithiothreitol (DTT) for approximately 30 minutes. Geobacillusstearothermophilus spores are then reacted with the reduced surface fora length of time from about 1 second to about 18 hours. Moreover, thespore is covalently bonded to the crosslinking agent which in turn iscovalently bonded to the amine functional group on the substrate.

EXAMPLE 1B Thiolation of a Spore

A spore solution of 10⁶ to 10⁹ cfu/mL is formulated with a PBS solution.A 120 μL aliquot of 20 mM SPDP is added to 0.5 mL of the sporesuspension. (The solutions can be scaled up as appropriate.) The SPDP isthen reacted with the spores for a period of 1 second to about 2 hours,with between 1 second and 30 minutes being preferred. The amine reactivegroup of the SPDP, N-hydroxysuccinimide (NHS) will react with availableamines in the proteins of the spore coat. The unreacted SPDP is thenremoved using typical methods such as dialysis, filtration, or both, orother means known to those skilled in the art. Following separation ofthe unreacted SPDP, the newly thiolated spores are brought in contactwith a surface that has been pre-modified with SPDP (following the samemethodology as used in other examples) and subsequently reduced. Thespore will then react with the surface creating a covalently linkagethrough a disulfide bound.

EXAMPLE 2 No Reducing Agent

Using a substrate with native hydroxyl functional groups (or a polymericsurface that has been treated with gas plasma to create hydroxylfunctionalities at the surface), the heterobifunctional reagent,N-(p-Maleimidophenyl) isocyanate (PMPI), is reacted to the surfacecontaining hydroxyl groups in a non-hydroxylic buffer at alkaline pH for30 minutes (a PMPI stock solution is made in either DMSO or DMF at anapproximately 10-fold molar excess over the concentration of hydroxylspresent at the surface). The isocyanate end group of the PMPI is thenreacted with the hydroxyl molecules on the surface of the substrate toform urethane linkages. The sulfhydryl groups present on the sporesGeobacillus stearothermophilus are then reacted with the maleimidefunctional end of the crosslinker at a neutral pH for a period of 2hours at room temperature. The heterobifunctional crosslinking reagentis covalently bonded to the spores and to the hydroxyl groups of thesubstrate.

EXAMPLE 3 No Reducing Agent

A substrate having either native double bonds, such as vinyl groups nearthe surface or a surface treated to create double bonds, is submerged ina sodium phosphate solution of pH 7-9 in the presence of a 10% solutionof 10 mM N-Succinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate(SANPAH) in either DMSO or DMF. The sample (substrate and solution) isexposed to UV light at a wavelength of 300-460 nm (desirably between 300nm-370 nm) for typically less than a minute. The nitrophenyl azide groupof the SANPAH forms a nitrene group which in turn initiates an additionreaction with the double bonds on the surface of the substrate. In aphosphate buffer at pH 7, a spore Geobacillus stearothermophilussuspension (concentration of 10⁷ cfu/ml) is brought in contact with themodified surface for a period of 60 minutes at room temperature. The NHSesters react with the primary amino groups to form stable amide bonds.The SANPAH crosslinking agent is covalently bonded to the spores and tothe vinyl group, located on the surface of the substrate.

In another embodiment of the present invention, a functionalized silanecoupling agent is utilized to add a functional group to the substratewith the functional group subsequently being directly attached to themicroorganism indicator with no intervening crosslinking reagent.Accordingly, no covalent bond is formed between the substrate functionalgroup and the microorganism such as a spore, but rather a physical orother type of non-covalent bond is formed. The functionalizing couplingagent can have the formula (FR)_(n)SiX_(4-n) where n equals from 1 to 3,and desirably from 1 to 3. R is an organic compound such as an alkylhaving from 1 to about 20 carbon atoms and desirably from 2 to about 18carbon atoms, and preferably from 3 to about 16, or an aromatic compoundhaving from 6 to about 20 carbon atoms, and desirably from 6 to about 15carbon atoms, or combinations thereof such as an alkylaryl, and anarylalkyl, and the like. X is a halide, such as a chloro or a bromogroup with chlorine being preferred, or an alkoxy group, OR¹, wherein R¹is an alkyl having from 1 to about 10 carbon atoms, and preferably from1 to 2 carbon atoms. Accordingly, a large number of silane couplingagents can be utilized and representative examples includepropyltrimethoxysilane, butyltrimethoxysilane, propyltriethoxysilane,butyltriethoxysilane, propyltrichlorosilane, propyltribromosilane,butyltrichlorosilane, butyltribromosilane, 11-hexadecyltrichlorosilane,15-pentadecenyltrichlorosilane, 11-bromoundecyltrichlorosilane,monochlorosilane, dichlorosilane, and the like. The silane couplingagent functional group, F, is a compound which can be attached to thebacteria, spore, etc. as through physical linkage. Suitable functionalgroups include thio compounds, amine compounds, carbonyl containingcompounds, bromine compounds, epoxy compounds, carboxyl containingcompounds, alkene compounds, and alkyne compounds, and the like, as wellas derivatives thereof. Examples of functionalized silane couplingagents thus include 3-aminopropyltrimethoxysilane,3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane,3-mercaptopropyltriethoxysilane, 3-aminopropyltrichlorosilane,3-mercaptopropyltrichlorosilane, and the like.

Since the various organic silanes generally have more than one X groupand typically 3, they can crosslink with hydroxyl groups whichinherently exist on some substrates such as glass and cellulose to forma two-dimensional mono-layer network of

groups wherein the oxygen atoms are derived from the hydroxyl groupbonded to the substrate surface. Naturally, a single type offunctionalized silane coupling agent can be utilized or a mixture of twoor more different types of functionalized silane coupling agents. Theend result is the creation of a high density of microorganism indicatorswhich are immobilized on the substrate surface.

The process of preparing the layer of the functionalized organosilanecoupling agents to the substrate comprises applying the silane compoundin a solvent which has been heated to about 60° C.-75° C. prior todeposition of the corresponding alkylsilane. Suitable solvents are anydry organic solvent, including aromatic hydrocarbons such as toluene,hexadecane, benzene, naphthalene, xylene, dry ketones such as acetoneand the like, with hexadecane being preferred.

The microorganism indicator such as spores can be physisorbed to thesurface through hydrophobic or electrostatic interactions between thefunctional molecules on the substrate surface and the proteins in thespore coat. The spores can also be chemisorbed to the surface using theamine groups present in the proteins.

EXAMPLE 4 Silane Linking Agent

Samples of a substrate material containing a closed adsorption cell or acrystallizing dish are prepared by cleaning with a solution of sulfuricacid and hydrogen peroxide to remove any organic impurities on thesurface. The substrate is then rinsed with copious amounts of water toremove any residual acid or peroxide.

The substrate is then soaked in a silane solution consisting of 1%hexadecyltrichlorosilane or 11-bromoundecyltrichlorosilane or15-pentadecenyltrichlorosilane in hexadecane for approximately 30seconds to 5 hours in a 60° C.-75° C. water bath. The substrate is thenremoved and rinsed with a non-polar solvent to remove residual silanemolecules.

To prepare the biological indicator, the functionalized substrate issubmerged, or dipped, into approximately 5-100 mLs of a spore solutionfor a time between about 1 second and about 18 hours, with the preferredtime between 1 second and 3 hours. Alternatively the functionalizedsubstrate can be inoculated, sprayed, or printed with the sporesuspensions. Following submersion, or dipping, in the spore solution,the substrate is left in ambient conditions (17° C.-25° C. and 1-30minutes) to dry. Once dry, the biological indicating article is thenrinsed with sterile water to remove any loosely attached spores. Thisrinse allows only the tenaciously attached spores to remain. Thebiological indicator is then dried again at ambient conditions andinspected to determine the number and distribution of the sporepopulation.

EXAMPLE 5 Silane Linking Agent

Samples of a glass substrate material are modified to contain an amineterminated surface thereon. The glass surfaces are cleaned using asolution of 30% hydrogen peroxide (35%) and 70% concentrated sulfuricacid. The amine functional surface is prepared by submerging the cleanedglass surface in a 1% solution of 3-aminopropyltrimethoxysilane in ananhydrous solvent, preferably hexadecane or acetone. The aminefunctional surface is soaked in a phosphate buffered saline solutioncontaining a 20 mM SPDP stock solution in either DMSO or EtOH for a timeof from about 30 minutes to about 60 minutes at a temperature between17° C.-25° C. Excess SPDP is then removed by rinsing in phosphatebuffer.

The substrate surface is then reduced using an acetate buffer solutioncontaining 25 mg/ml dithiothreitol (DTT). The surface is soaked in theacetate/DTT solution for 30 minutes at a temperature of 17° C.-25° C.The surfaces are then rinsed with copious amounts of acetate buffer toremove any DTT.

To prepare the biological indicating article, the substrate with theheterobifunctional agents attached is submerged, into an appropriatevolume of a spore solution for a time between about 30 seconds and about20 hours. Following submersion, or dipping, in the spore solution, thesubstrate is left in ambient conditions [17° C.-25° C.] to dry.

EXAMPLE 6

In yet another embodiment, hydroxyl functionalized surfaces, such asborosilicate glass, can be used to prepare the biological indicator. Thefunctionalized substrate is submerged, or dipped, into approximately5-100 mLs of a spore solution for a time between about 1 second andabout 24 hours, with the preferred time between 1 second and 3 hours.Following submersion, or dipping, in the spore solution, the substrateis left in ambient conditions (17-25° C. and 1-30 minutes) to dry. Oncedry, the biological indicating article is then rinsed with sterile waterto remove any loosely attached spores. The rinse allows only thetenaciously attached spores to remain. The biological indicator is thendried again at ambient conditions and visually inspected to determinedistribution of the spore population and the number of spores on asurface. A uniform spore population is obtained wherein the standard ofdeviation is approximately 25% or less.

While in accordance with the patent statutes, the best mode andpreferred embodiment have been set forth, the scope of the invention isnot limited thereto, but rather by the scope of the attached claims.

1. A biological indicator for monitoring exposure to and effectivenessof a sterilization or disinfection process, comprising: a substrate andoptionally a surface layer residing on said substrate, said substrate orsaid optional surface layer having an exposed surface thereon; afunctionalized silane coupling agent chemically bound to said exposedsurface; and a microorganism indicator non-covalently bonded tofunctional groups of said functionalized silane, said microorganismindicator comprising an endospore, a fungi, a mycobacteria, a vegetativebacteria, or a protozoa, or combinations thereof.
 2. A biologicalindicator according to claim 1, wherein said silane has functionalgroups comprising a thio, an amine, a carbonyl, a bromine, an epoxy, acarboxyl, or an alkene or alkyne, or combinations thereof.
 3. Abiological indicator according to claim 2, wherein said optional surfacelayer is not present, wherein said microorganism endospore indicatorcomprises Geobacillus stearothermophilus, Bacillus subtilis, Bacillussubtilis globigii, Clostridium sporogenes, Bacillus cereus, or Bacilluscirculans, or combinations thereof; wherein said microorganism fungiindicator comprises Aspergillus niger, Candida albicans, Trichophytonmentagrophytes, or Wangiella dermatitis, or combinations thereof;wherein said microorganism mycobacteria indicator comprisesMycobacterium chelonae, Mycobacterium gordonae, Mycobacterium smegmatis,or Mycobacterium terrae, or combinations thereof; wherein saidvegetative bacteria indicator comprises Aeromonas hydrophila,Enterococcus faecalis, Streptococcus faecalis, Enterococcus facecium,Streptococcus pyrogenes, Escherichia coli, Klebsiella pneumoniae,Legionella pneumophila, Methylobacterium, Pseudomonas aeruginosa,Salmonella choleraesuis, Helicobacter pylori, Staphylococcus aureus,Staphylococcus epidermidis, or Stenotrophomonas maltophilia, orcombinations thereof, and wherein said microorganism protozoa indicatorcomprises Giardia lamblia or Cryptosporidium parvum, or combinationsthereof.
 4. A biological indicator according to claim 3, wherein saidfunctional groups comprise an amine or a hydroxyl; wherein saidmicroorganism endospore indicator has a sulfur end group thereon andcomprises Geobacillus stearothermophilus or Bacillus subtilis; whereinthe concentration of said microorganism endospore indicators is fromabout 10⁴ to about 10⁷ cfu/biological indicator; and wherein thedistribution of said microorganism endospore indicators on saidsubstrate is uniform, wherein the standard deviation of the distributionis about 25% or less based on one unit surface area of said substrate ascompared to another unit surface area of said substrate.